Deepak Choudhury

Cell culture on MEMS platforms: a review.

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.

Galactosylated cellulosic sponge for multi-well drug safety testing

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.

Exploitation of physical and chemical constraints for three-dimensional microtissue construction in microfluidics

There are a plethora of approaches to construct microtissues as building blocks for the repair and regeneration of larger and complex tissues. Here we focus on various physical and chemical trapping methods for engineering three-dimensional microtissue constructs in microfluidic systems that recapitulate the in vivo tissue microstructures and functions. Advances in these in vitro tissue models have enabled various applications, including drug screening, disease or injury models, and cell-based biosensors. The future would see strides toward the mesoscale control of even finer tissue microstructures and the scaling of various designs for high throughput applications. These tools and knowledge will establish the foundation for precision engineering of complex tissues of the internal organs for biomedical applications.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.

Scalable cell alignment on optical media substrates

Cell alignment by underlying topographical cues has been shown to affect important biological processes such as differentiation and functional maturation in vitro. However, the routine use of cell culture substrates with micro- or nano-topographies, such as grooves, is currently hampered by the high cost and specialized facilities required to produce these substrates. Here we present cost-effective commercially available optical media as substrates for aligning cells in culture. These optical media, including CD-R, DVD-R and optical grating, allow different cell types to attach and grow well on them. The physical dimension of the grooves in these optical media allowed cells to be aligned in confluent cell culture with maximal cell-cell interaction and these cell alignment affect the morphology and differentiation of cardiac (H9C2), skeletal muscle (C2C12) and neuronal (PC12) cell lines. The optical media is amenable to various chemical modifications with fibronectin, laminin and gelatin for culturing different cell types. These low-cost commercially available optical media can serve as scalable substrates for research or drug safety screening applications in industry scales.

Microfluidic platforms for modeling biological barriers in the circulatory system

Microfluidic platforms have recently become popular as in vitro models because of their superiority in recapitulating microenvironments compared with conventional in vitro models. By providing various biochemical and biomechanical cues, healthy and diseased models at the organ level can be applied to disease progression and treatment studies. Microfluidic technologies are especially suitable for modeling biological barriers because the flow in the microchannels mimics the blood flow and body fluids at the interfaces of crucial organs, such as lung, intestine, liver, kidney, brain, and skin. These barriers have similar structures and can be studied with similar approaches for the testing of pharmaceutical compounds. Here, we review recent developments in microfluidic platforms for modeling biological barriers in the circulatory system.

Organ-Derived Decellularized Extracellular Matrix: A Game Changer for Bioink Manufacturing?

The extracellular matrix (ECM) comprises a complex milieu of proteins and other growth factors that provide mechanical, biophysical, and biochemical cues to cells. The ECM is organ specific, and its detailed composition varies across organs. Bioinks are material formulations and biological molecules or cells processed during a bioprinting process. Organ-derived decellularized ECM (dECM) bioinks have emerged as arguably the most biomimetic bioinks. Here, we review bioinks derived from different decellularized organs, the techniques used to obtain these bioinks, and the characterization methods used to evaluate their quality. We emphasize that obtaining a good-quality bioink depends on the choice of organ, animal, and decellularization method. Finally, we explore potential large-scale applications of bioinks and challenges in manufacturing such bioinks.

Neuromodulatory circuit effects on Drosophila feeding behaviour and metabolism

Animals have evolved to maintain homeostasis in a changing external environment by adapting their internal metabolism and feeding behaviour. Metabolism and behaviour are coordinated by neuromodulation; a number of the implicated neuromodulatory systems are homologous between mammals and the vinegar fly, an important neurogenetic model. We investigated whether silencing fly neuromodulatory networks would elicit coordinated changes in feeding, behavioural activity and metabolism. We employed transgenic lines that allowed us to inhibit broad cellular sets of the dopaminergic, serotonergic, octopaminergic, tyraminergic and neuropeptide F systems. The genetically-manipulated animals were assessed for changes in their overt behavioural responses and metabolism by monitoring eleven parameters: activity; climbing ability; individual feeding; group feeding; food discovery; both fed and starved respiration; fed and starved lipid content; and fed/starved body weight. The results from these 55 experiments indicate that individual neuromodulatory system effects on feeding behaviour, motor activity and metabolism are dissociated.

On chip two-photon metabolic imaging for drug toxicity testing

We have developed a microfluidic system suitable to be incorporated with a metabolic imaging method to monitor the drug response of cells cultured on a chip. The cells were perfusion-cultured to mimic the blood flow in vivo. Label-free optical measurements and imaging of nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence intensity and morphological changes were evaluated non-invasively. Drug responses calculated using redox ratio imaging were compared with the drug toxicity testing results obtained with a traditional well-plate system. We found that our method can accurately monitor the cell viability and drug response and that the IC50 value obtained from imaging analysis was sensitive and comparable with a commonly used cell viability assay: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) assay. Our method could serve as a fast, non-invasive, and reliable way for drug screening and toxicity testing as well as enabling real-time monitoring of in vitro cultured cells.

Gratings on a Dish: A Scalable Cell Alignment Substrate on Optical Media

Cell alignment by underlying topographical cues has been shown to affect important biological processes such as differentiation and functional maturation in vitro. However, the routine use of cell culture substrates with micro/nano-topographies is currently hampered by the high cost and specialized facilities required to produce these substrates. Here we present commercially available optical media as substrates for aligning cells in culture. These optical media, including CD-R, DVD-R and optical grating, allow different cell types to attach, align and grow on them. This cytoskeletal reorganization enhanced the differentiation of cardiac (H9C2), and skeletal muscle (C2C12) cell lines. These low-cost commercially available optical media can serve as scalable substrates for research or drug safety screening applications.Copyright